The mammalian intestinal mucosa is in a constant state of renewal that is characterized by active proliferation of stem cells localized in the crypts, progression of these cells up the crypt villus axis with cessation of proliferation, and subsequent differentiation. A delicate and precise balance of proliferation -> differentiation -> apoptosis must exist in the gut mucosa to maintain this normal intestinal homeostasis. Surgical resection of the small intestine results in an adaptive hyperplasia, whereas the inability to provide enteral nutrients, as occurs in certain post-operative or trauma patients, results in atrophy. The major focus of our group has been the analysis of mechanisms regulating intestinal proliferation, differentiation, adaptation, and apoptosis. With the support of this grant, we have made great strides in our better understanding of the molecular mechanisms regulating intestinal homeostasis. We have shown that stimulation of PI3K/Akt increases normal intestinal proliferation. Our most recent findings have identified novel and differential roles for the mammalian target of rapamycin (mTOR) protein complex, a major downstream effector of PI3K, in intestinal proliferation and differentiation and intestinal cell survival. In preliminary findings, we have shown that inhibition of mTORC2, but not mTORC1, enhances intestinal cell differentiation, suggesting differential effects for these proteins in the gut. Also, we have recently described the molecular mechanisms contributing to glutamine-mediated intestinal cell survival and proliferation; the PI3K/mTOR pathway plays an important protective role to limit apoptosis associated with cellular stress. Finally, we have demonstrated a novel role for the mTOR pathway in intestinal cell autophagy following glutamine deprivation. Based on our recent published and preliminary findings, we have refined our central hypothesis to state that intestinal cell proliferation and differentiation are regulated by signaling mechanisms involving the PI3K/mTOR pathway acting on critical downstream effector proteins; novel and differential effects of mTOR and Akt components exist that further add to the complex, but highly regimented, regulation of intestinal homeostasis. To examine this hypothesis, we have planned experiments with the following Specific Aims: 1) to determine the differential effects of mTOR complexes on intestinal cell homeostasis, 2) to delineate the signaling proteins that mediate the effects of mTOR in the intestine, and 3) to define the role of mTOR/mTOR complexes in intestinal cell growth in vivo. The studies in the current proposal represent direct extensions of our previous findings, and are designed to define in a systematic fashion the molecular mechanisms and signaling events regulating the processes of intestinal proliferation, differentiation, and adaptation. To achieve our goals, we have assembled a multidisciplinary, diverse, and highly collaborative team of investigators who each bring unique expertise and background to the project. PUBLIC HEALTH RELEVANCE: The intestinal mucosa is in a constant state of growth and renewal; the molecular pathways contributing to this critical function are poorly understood. Our recent findings have identified novel roles for components of the PI3K/Akt/mTOR signaling pathway in intestinal homeostasis. Delineating the cellular factors and signaling pathways regulating these processes is crucial to our understanding of not only normal gut development and maturation, but also the effects of surgical resection and stress on intestinal adaptation and survival.